CN115011645B - 吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用 - Google Patents
吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体公开了吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用，本发明吡啶甲酸来源于溶杆菌(Lysobacter sp.)OH23，并从该菌株的培养液中分离纯化吡啶甲酸，利用平板筛选法检测吡啶甲酸对黄单胞菌具有特异性的拮抗活性，另外，从培养液中分离纯化的方法简单，快捷，高效，相比于化学合成对环境更友好，其针对水稻黄单胞菌具有特异的抗菌活性，为开发防治水稻黄单胞菌病害的新型生物农药提供了有利的基础。
Description
技术领域
本发明属于生物技术领域，具体涉及吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用。
背景技术
黄单胞菌(Xanthomonas)是一类重要的植物病原细菌，在世界范围引起几十种农作物的灾难性病害。水稻是我国主要粮食作物之一，由水稻黄单胞菌两个致病变种水稻白叶枯病菌(X.oryzae pv.oryzae)和条斑病菌(X.oryzae pv.oryzicola)引起的水稻白叶枯病和条斑病严重制约着水稻的产量和质量。目前，针对这两个病害的化学防治以保护性农药为主，长期施用引致的病菌抗药性已经成为食品质量安全和生态安全关注的焦点。因此，寻找新的生防微生物或其代谢产物并开发为生态安全、环境友好的生物农药将是今后病害绿色防治的发展方向。
吡啶甲酸(Picolinic acid)是犬尿氨酸途径中L-色氨酸的终产物，它经常在各种各样的生物介质中，如细胞培养上清液、血清和人奶。吡啶甲酸也可以通过2-氨基苯酚，硝基苯，邻苯二酚，邻氨基苯甲酸和3-羟基对氨基苯甲酸等物质的微生物降解产生。另外，吡啶甲酸是一些有机合成药物、除草剂、杀菌剂的重要中间体。有研究表明吡啶甲酸能够抑制小鼠肾细胞生长、T细胞增值，并且具有抗微生物活性，但还未见吡啶甲酸能够抑制水稻黄单胞菌生长的报道。
溶杆菌(Lysobacter)属于γ-变形菌纲的黄单胞菌科，因其可产生大量胞外酶及活性次生代谢产物，并且环境友好，正作为新的农用抗生素来源受到越来越多的关注。申请人从水稻根际土壤分离到一株溶杆菌(Lysobacter sp.)OH23，并发现该菌对黄单胞病菌具有高效、特异性的拮抗活性。因此，如何利用溶杆菌OH23在生物农药上进行应用，值得大家继续探究。
发明内容
针对现有技术的不足，本发明的目的在于提供吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用，本发明吡啶甲酸来源于溶杆菌(Lysobacter sp.)OH23，并从该菌株的培养液中分离纯化吡啶甲酸，利用平板筛选法检测吡啶甲酸对黄单胞菌具有特异性的拮抗活性，另外，从培养液中分离纯化的方法简单、快捷、高效，为开发防治水稻黄单胞菌病害的新型生物农药提供基础。
为解决现有技术问题，本发明采取的技术方案为：
一种吡啶甲酸，所述吡啶甲酸来源于溶杆菌(Lysobacter sp.)OH23的培养液，所述所述溶杆菌(Lysobacter sp.)OH23保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号：CGMCC No.13677，保藏日期：2017年2月23日，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
上述吡啶甲酸来源于溶杆菌(Lysobacter sp.)OH23的培养液的分离纯化方法，包括以下步骤：
步骤1，溶杆菌(Lysobacter sp.)OH23于NB培养基培养获得中发酵，离心取上清液；
步骤2，用HPLC分离纯化上清液，流动相为含0.06N H2SO4、pH为5的水，流速为0.8-1.2mL/min，30min，60℃，紫外吸收波长为254nm，即得吡啶甲酸。
作为改进的是，步骤1中发酵的方法为：挑取(Lysobacter sp.)OH23菌落于NB培养基中培养得种子液，种子液以1％体积转接至NB培养基中，28℃、180rpm培养48h，再将发酵液在10000rpm下离心20min获得上清液。
作为改进的是，步骤2中分离纯化所用的色谱柱为C-18(250×9.4mm)，
上述吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用。
有益效果：
与现有技术相比，本发明吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用，通过培养溶杆菌(Lysobacter sp.)OH23，确定溶杆菌OH23具有生产吡啶甲酸的功能，建立高效液相色谱法分离纯化吡啶甲酸的方法，该方法简便、高效，相比于化学合成对环境更友好，测定其针对黄单胞菌的特异抗菌活性，对黄单胞菌引起的作物病害防治具有重要意义。利用平板筛选法检测吡啶甲酸的抑菌活性，结果显示吡啶甲酸对黄单胞菌具有特异性的拮抗活性，而对其它病原细菌的抑制活性较弱。并且，温室试验表明含有吡啶甲酸的OH23发酵液对水稻白叶枯病具有良好的防治效果，可减少病斑长度36.7％。
附图说明
图1为溶杆菌OH23上清HPLC色谱图；
图2为纯化的吡啶甲酸HPLC色谱图；
图3为吡啶甲酸质谱图；
图4为温室中溶杆菌(Lysobacter sp.)OH23发酵液对水稻白叶枯病的防治效果。
具体实施方式
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
实施例1溶杆菌OH23菌种活化及发酵上清液制备
将溶杆菌OH23菌种从-80℃取出划线于NB固体培养基，28℃培养48h，挑取单菌落于NB液体培养基，28℃、180rpm培养48h得到种子液。将种子液以1％接种量接种于NB液体培养基，28℃、180rpm培养48h，得到发酵液。发酵液经10000rpm离心20min，获得发酵液上清。
实施例2溶杆菌OH23发酵上清中吡啶甲酸的分离纯化
上清液过滤后，经过HPLC分离纯化，制备条件：色谱柱型号为C-18(250×9.4mm)，流动相为含0.06N H2SO4、pH为5的水，流速为1.2mL/min，30min，柱温60℃，紫外检测波长为254nm。保留时间为17.2min的色谱峰为吡啶甲酸，HPLC的结果如图1，纯化的吡啶甲酸HPLC检测如图2，分子量[M+H]+值如图3。
实施例3溶杆菌OH23发酵上清中纯化的吡啶甲酸的抗菌活性
将吡啶甲酸加到NB培养基中，再将含吡啶甲酸的培养基倒入培养皿中，形成吡啶甲酸终浓度分别为：0、50μg/mL、100μg/mL、250μg/mL、500μg/mL、750μg/mL、1000μg/mL、1250μg/mL、1500μg/mL、2000μg/mL的含药培养皿。各类细菌培养至OD1.0后，取4μL滴于含药培养皿上，28℃培养48h。观察结果，并记录入表1中。
表1溶杆菌OH23产生的吡啶甲酸的抑菌效果
观察结果，结果显示吡啶甲酸对水稻黄单胞菌的MIC≤50μg/mL，对其它黄单胞菌的MIC≤100μg/mL，对于其它细菌的拮抗活性较弱(表1)。
通过培养溶杆菌(Lysobacter sp.)OH23，确定溶杆菌OH23具有生产吡啶甲酸的功能，建立高效液相色谱法分离纯化吡啶甲酸的方法，该方法简便、高效，相比于化学合成对环境更友好，测定其针对黄单胞菌的特异抗菌活性，对黄单胞菌引起的作物病害防治具有重要意义。利用平板法检测吡啶甲酸的抑菌活性，结果显示吡啶甲酸对黄单胞菌具有特异性的拮抗活性，而对其它病原细菌的抑制活性较弱。
实施例4 OH23发酵液在温室中对水稻白叶枯病的防治效果
选取生长期7周的水稻植株，每个处理6小盆水稻作为重复，喷施含有吡啶甲酸的OH23发酵液100mL。一天后，通过剪叶法进行水稻白叶枯病菌的接种。
具体方法为：将水稻白叶枯病菌培养至对数期，OD600调至1.0，利用长口手术剪蘸取菌液在距离水稻叶尖2cm处剪叶接种。接种后，置于人工温室培养，设定温度为28℃，相对湿度为70％。14天后，测量病斑长度，结果显示喷施OH23发酵液的水稻植株病斑长度较对照减少36.7％(图4)。
以上结果表明OH23发酵液在温室中对水稻白叶枯病具有较好的防治效果。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (3)
1.吡啶甲酸在制备防治黄单胞菌病害的生物农药上的应用，其特征在于，所述吡啶甲酸来源于溶杆菌(Lysobacter sp.) OH23的培养液，所述吡啶甲酸的分离纯化方法，包括以下步骤：步骤1，溶杆菌(Lysobacter sp.) OH23于NB培养基培养获得中发酵，离心取上清液；步骤2，用HPLC分离纯化上清液，流动相为含0.06N H2SO4 、pH为5的水，流速为0.8-1.2mL/min，30min，60℃，紫外吸收波长为254nm，即得吡啶甲酸；其中，所述溶杆菌(Lysobacter sp.) OH23保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号：CGMCC No. 13677，保藏日期：2017年2月23日，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
2.根据权利要求1所述的应用，其特征在于，步骤1中发酵的方法为：挑取(Lysobactersp.) OH23菌落于NB培养基中培养得种子液，种子液以1%体积转接至NB培养基中，28℃、180rpm培养48h，再将发酵液在10000rpm下离心20min获得上清液。
3.根据权利要求1所述的应用，其特征在于，步骤2中分离纯化所用的色谱柱为C-18。
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